Neuron Specific Promoters for Aav Gene Transfer

This application claims priority under 35 U.S.C. $119 (e) to U.S. Provisional Patent Application Ser. No. 63/496,440, filed Apr. 17, 2023, and entitled “NEURON SPECIFIC PROMOTERS FOR AAV GENE TRANSFER,” which is herein incorporated by reference in its entirety for all purposes.

Recombinant adeno-associated viral (rAAV) vectors are currently considered the leading platform for in vivo gene therapy. However, the packaging capacity of AAV is limited to <5 kb, which excludes larger therapeutic genes from conventional vector designs. Many efforts have been developed to overcome this limitation, such as split rAAVs and fragmented rAAV strategies.

Aspects of the disclosure relate to compositions and methods for expressing heterologous gene products (e.g., proteins, such as therapeutic proteins, and functional RNAs such as interfering nucleic acids) in target cells of a subject. In some embodiments, the target cells are cells of the nervous system (e.g., CNS or PNS) of a subject. The disclosure is based, in part, on variants of SMN1 promoters that are beneficial for packaging and expressing larger transgenes. In some embodiments, the promoters are truncated SMN1 promoters (e.g., promoters having nucleotide sequences that are shortened relative to a native SMN1 promoter, for example a human SMN1 promoter, such as those set forth in SEQ ID NOs: 5 and 6).

Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising a human SMN1 promoter variant comprising a nucleic acid sequence that is at least 60% identical to a nucleic acid sequence set forth in any one of SEQ ID NOs: 1-3 and does not comprise the sequence set forth in SEQ ID NO: 5 or 6.

In some embodiments, a human SMN1 promoter variant comprises a nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, a human SMN1 promoter variant comprises a nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, a human SMN1 promoter variant comprises a nucleic acid sequence set forth in SEQ ID NO: 3.

In some embodiments, an isolated nucleic acid further comprises a protein coding nucleic acid sequence operably linked to the promoter. In some embodiments, the protein coding nucleic acid encodes a therapeutic protein. In some embodiments, the therapeutic protein is a Survival Motor Neuron (SMN) protein.

In some embodiments, an isolated nucleic acid comprises an interfering nucleic acid sequence operably linked to the human SMN1 promoter. In some embodiments, the interfering nucleic acid is a dsRNA, SIRNA, shRNA, miRNA, artificial miRNA (ami-RNA), or RNA aptamer.

In some embodiments, an isolated nucleic acid further comprises a polyA region positioned 3′ relative to the nucleic acid sequence encoding the protein or the interfering nucleic acid.

In some embodiments, an isolated nucleic acid further comprises one or more miRNA binding sites.

In some embodiments, an isolated nucleic acid further comprises adeno-associated virus (AAV) inverted terminal repeats (TTRs).

In some aspects, the disclosure provides vector comprising an isolated nucleic acid as described herein.

In some aspects, the disclosure provides a recombinant AAV (rAAV) comprising an isolated nucleic acid as described herein: and at least one AAV capsid protein.

In some embodiments, at least one capsid protein has a serotype selected from an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 serotype. In some embodiments, at least one capsid protein is an AAV9 capsid protein.

In some embodiments, an rAAV is a self-complementary AAV (scAAV). In some embodiments, an rAAV is a self-complementary AAV9 (scAAV9).

In some embodiments, a protein coding nucleic acid sequence encodes a therapeutic protein. In some embodiments, a protein coding nucleic acid sequence encodes a Survival Motor Neuron (SMN) protein.

In some aspects, the disclosure provides a method of expressing a gene product in a subject, the method comprising administering an isolated nucleic acid or rAAV as described herein to the subject.

In some embodiments, a subject is a mammal. In some embodiments, a subject is a human.

In some embodiments, administration to a subject comprises injection. In some embodiments, injection comprises direct injection to the CNS of the subject or systemic injection.

In some embodiments, a gene product is a therapeutic protein encoded by an isolated nucleic acid or rAAV.

In some embodiments, administration of an isolated nucleic acid or rAAV results in expression of a gene product in CNS cells or peripheral nervous system (PNS) cells of a subject. In some embodiments. CNS cells comprise spinal cord neurons.

Aspects of the disclosure relate to compositions and methods for delivery of certain gene products (e.g., proteins, nucleic acids, etc.) to a subject, e.g., to the central nervous system (CNS) of a subject. The disclosure is based, in part, on expression cassettes (e.g., isolated nucleic acids) comprising a nucleic acid sequence operably linked to a human SMN1 promoter or a variant of a human SMN1 promoter, for example variants that are truncated relative to a native SMN1 promoter, for example a human SMN1 promoter (e.g., promoters comprising the sequence set forth in SEQ ID NO: 5 or 6). In some embodiments, a human SMN1 promoter is capable of driving physiological expression level of SMN in certain cells of a subject, for example CNS cells (e.g., spinal cord neurons) of a human subject. The human SMN1 promoter variants may be used as a portion of an isolated nucleic acid (e.g., DNA, plasmid vector, rAAV vector, etc.) or as a portion of a viral particle, for example a recombinant adeno-associated virus (rAAV) or recombinant lentiviral particle.

Aspects of the disclosure also provide methods for delivering a transgene (e.g., a gene product, for example a therapeutic protein and/or inhibitory nucleic acid) to a target tissue of a subject, such as CNS tissue. The disclosure is based, in part, on human SMN1 promoter variants that are truncated (e.g., shortened) relative to a native human SMN1 promoter and allow for efficient transgene expression in certain cells (e.g., CNS cells of a subject) while reducing the promoter size. In some embodiments, reduced promoter size is useful for incorporating such promoters into vectors having limited transgene capacity, for example rAAV vectors. In some embodiments, delivery of a transgene (e.g., SMN1) to CNS cells of a subject is useful for treating central nervous system (CNS) diseases.

Aspects of the disclosure relate to nucleic acids and vectors, for example viral vectors, that comprise a human SMN1 promoter or a variant of a human SMN 1 promoter (e.g., as described in any one of SEQ ID NOs: 1-3). A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively linked.” “operatively positioned.” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

In some embodiments, the promoter (e.g., human SMN1 promoter) imparts tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.,) are well known in the art.

In some embodiments, the human SMN1 promoter preferentially drives expression of a gene product encoded by a nucleic acid in certain tissues. In some embodiments, the disclosure provides a nucleic acid comprising a tissue-specific human SMN1 promoter (e.g., a human SMN1 promoter or a human SMN1 promoter variant operably linked to a nucleic acid sequence (e.g., a transgene encoding a protein, such as a therapeutic protein, e.g., SMN1). As used herein, “tissue-specific promoter” refers to a promoter that preferentially regulates (e.g., drives or up-regulates) gene expression in a particular cell type relative to other cell types. A cell-type-specific promoter can be specific for any cell type, such as central nervous system (CNS) cells, liver cells (e.g., hepatocytes), heart cells, muscle cells, etc. In some embodiments, a tissue-specific promoter is a CNS-tissue-specific, PNS-tissue-specific, or cell-specific promoter. In some embodiments, the human SMN1 promoter (or human SMN1 promoter variant) is tissue-specific to, e.g., kidney cells, neuroblasts, spinal cord neurons, brain cells, liver cells, heart cells, and/or muscle cells.

Aspects of the disclosure relate to isolated nucleic acids and rAAV vectors comprising a nucleic acid sequence encoding a gene product operably linked to its native promoter. In some embodiments a native promoter comprises a human SMN1 promoter, or a variant thereof (e.g., as described in any one of SEQ ID NOs: 1-3). In some embodiments, a human SMN1 promoter variant comprises a nucleic acid sequence that has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 945%, at least 95%, at least 969%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence set forth in one of SEQ ID NOs: 1-3. A native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression (e.g., express physiological levels of a gene product, for example a therapeutic protein such as SMN, expression in the appropriate cell types). The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. Without wishing to be bound by any theory, use of a human SMN1 promoter in isolated nucleic acids and rAAV vectors described herein regulates expression of gene products from the vectors, and reduces toxicity, for example cytotoxicity or hepatotoxicity, in a subject relative to expression of the gene products from isolated nucleic acids and rAAV vectors comprising other promoters, for example CMV promoter, chicken-beta actin (CBA) promoter, CB6 promoter, etc. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, and/or Kozak consensus sequences may also be used to mimic the native expression.

In some embodiments, the nucleic acid sequence encoding SMN is operably linked to a human SMN1 promoter. In certain embodiments, a human SMN1 promoter is configured to drive physiological expression level of a gene product (e.g., a therapeutic protein or an interfering RNA). In some embodiments, the human SMN1 promoter comprises a sequence that is set forth in one of SEQ ID NOs: 1-3. In some embodiments, the human SMN1 promoter consists of a sequence that is set forth in one of SEQ ID NOs: 1-3.

In some embodiments, the nucleic acid comprises the sequence set forth in one of SEQ ID NOs: 1.3. In some embodiments, the nucleic acid has a sequence having at least 10% sequence identity, at least 20% sequence identity, at least 30% sequence identity, at least 40% sequence identity, at least 50% sequence identity, at least 55% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 1009% sequence identity with one of SEQ ID NOs: 1-3. In certain embodiments, the nucleic acid has a sequence having 100% sequence identity with one of SEQ ID NOs: 1-3. In certain embodiments, a human SMN1 promoter variant has unexpectedly beneficial performance in spinal cord neurons, neurons and/or muscle cells.

In some embodiments, the nucleic acid has a human SMN1 promoter variant sequence that is truncated relative to SEQ ID NO: 5 or 6 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments, the nucleic acid has a human SMN1 promoter variant sequence that is truncated relative to SEQ ID NO: S or 6 by at least 20, 50, 75, 100, 140, 148, 200, 240, 250, 300, 350, 400, 450, 500, or more nucleotides.

In some embodiments, the human SMN1 promoter has a length of at least 61 base pairs, at least 65 base pairs, at least 70 base pairs, at least 75 base pairs, at least 80 base pairs, at least 85 base pairs, or at least 90 base pairs. In some embodiments, the human SMN1 promoter has a length of at most 303 base pairs, at most 300 base pairs, at most 250 base pairs, at most 200 base pairs, at most 155 base pairs, at most 150 base pairs, at most 145 base pairs, at most 140 base pairs, at most 135 base pairs, at most 130 base pairs, at most 125 base pairs, at most 120 base pairs, at most 115 base pairs, at most 110 base pairs, at most 105 base pairs, at most 100 base pairs, at most 95 base pairs. Combinations of the above-referenced ranges are also possible (e.g., at least 61 base pairs and at most 303 base pairs, at least 61 base pairs and at most 155 base pairs). Other ranges are also possible.

The terms “percent identity.” sequence identity. “% identity,” % sequence identity.” and % identical,” as they may be interchangeably used herein, refer to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid).

Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 909%, at least 959%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk. A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press. 1987; Computer Analysis of Sequence Data, Part I. Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey. 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman. D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, OCG program package, Devereux. J., et al., Nucleic Acids Research, 12 (1), 387 (1984)). BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

When a percent identity is stated, or a range thereof (e.g., at least, more than, etc.), unless otherwise specified, the endpoints shall be inclusive and the range (e.g., at least 70% identity) shall include all ranges within the cited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 759%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 826%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 889%, at least 89%, at least. 909%, at least 919%, at least 92%, at least 939%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5% at least 98%, at least 98.59%, at least 99%, at least 99.5%, at least 99.69%, at least 99.79%, at least 99.89%, at least 99.9% identity) and all increments thereof (e.g., tenths of a percent (e.g., 0.1%), hundredths of a percent (e.g., 0.01%), etc.).

Aspects of the disclosure relate to nucleic acids, for example isolated nucleic acids. A “nucleic acid” sequence refers to a DNA or RNA sequence. In some embodiments, proteins and nucleic acids of the disclosure are isolated. As used herein, the term “isolated” means artificially produced. As used herein, with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5′ and 3′ restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term “isolated” refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and/or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats (ITRs). The transgene may comprise a region encoding, for example, a protein and/or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure.

Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See. e.g., texts such as Sambrook ci al., “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol, 70:520 532 (1996). An example of such a molecule employed in the disclosure is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, an isolated nucleic acid encoding a transgene is flanked by AAV ITRs (e.g., in the orientation 5′-ITR-transgene-ITR-3′). In some embodiments, the AAV TTRs are AAV2 TTRs. In some embodiments, at least one AAV ITR is a truncated AAV ITR (e.g., a mutant ITR, also referred to as an mTR), for example a ΔITR as described, for example by McCarty (2008) Molecular Therapy 16 (10): 1648-1656.

In addition to the major elements identified above for the recombinant AAV vector, the vector also includes conventional control elements which are operably linked with elements of the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the vector or infected with the virus produced by the disclosure. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence; sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein of polypeptide. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame. In some embodiments, operably linked coding sequences yield a fusion protein.

A region comprising a transgene (e.g., a transgene encoding a gene product, for example SMN protein, etc.) may be positioned at any suitable location of the isolated nucleic acid that will enable expression of the at least one transgene, the selectable marker protein, or reporter protein.

For nucleic acids encoding proteins, a polyadenylation sequence generally is inserted following the transgene sequences and before the 3′ AAV ITR sequence. A rAAV construct useful in the disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence. In some embodiments, an intron is a non-native intron or synthetic intron (e.g., a MBL intron). Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein that contains more than one polypeptide chains. Selection of these and other common vector elements are conventional, and many such sequences are available [see, e.g., Sambrook et al., and references cited therein at, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4:928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208: de Felipe, P et al., Human Gene Therapy, 2000; 11:1921-1931.; and Klump, H et al., Gene Therapy, 2001; 8:811-817).

In some embodiments, one or more bindings sites for one or more of miRNAs are incorporated in a transgene of a rAAV vector, to inhibit the expression of the transgene in one or more tissues of an subject harboring the transgene. The skilled artisan will appreciate that binding sites may be selected to control the expression of a transgene in a tissue specific manner. For example, binding sites for the liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. The target sites in the mRNA may be in the 5′ UTK, the 3′ UTR or in the coding region. Typically, the target site is in the 3′ UTR of the mRNA. Furthermore, the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may result in the cooperative action of multiple RISC's and provide highly efficient inhibition of expression. The target site sequence may comprise a total of 5-100, 10-60, or more nucleotides. The target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site.

Aspects of the disclosure relate to isolated nucleic acids comprising a human SMN1 promoter or human SMN1 promoter variant operably linked to a nucleic acid sequence encoding a gene product. A gene product may be a peptide, protein, nucleic acid, or a combination thereof. In some embodiments, the nucleic acid gene product is a therapeutic functional RNA (e.g., an interfering RNA, such as dsRNA, siRNA, miRNA, artificial miRNA (ami-RNA), RNA aptamer, etc.). In some embodiments, a nucleic acid comprising a human SMN1 promoter or human SMN1 promoter variant operably linked to a nucleic acid sequence encoding a gene product is referred to as a transgene. In some embodiments, a transgene further comprises one or more additional regulatory sequences, such as an enhancer, polyA region, etc.

For example, one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal. In another example, the transgene encodes a therapeutic protein or therapeutic functional RNA. In another example, the transgene encodes a transmembrane protein. In another example, the transgene encodes a secreted protein. In another example, the transgene encodes a protein or functional RNA that is intended to be used for research purposes, e.g., to create a somatic transgenic animal model harboring the transgene. e.g., to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of disease. Appropriate transgene coding sequences will be apparent to the skilled artisan.

Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, Ovalbumin (OVA) and others well known in the art. When associated with regulatory elements which drive their expression, the reporter sequences, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for β-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer. Such reporters can, for example, be useful in verifying the tissue-specific targeting capabilities and tissue specific promoter regulatory activity of an rAAV.

In some aspects, the disclosure provides vectors (e.g., TAAV vectors, lentiviral (LV) vectors, adenoviral vectors, plasmids, cosmids, etc.) for use in methods of preventing of treating one or more genetic deficiencies or dysfunctions in a mammal, such as for example, a polypeptide deficiency or polypeptide excess in a mammal, and particularly for treating or reducing the severity or extent of deficiency in a human manifesting one or more of the disorders linked to a deficiency in such polypeptides in cells and tissues. The method involves administration of a vector that encodes one or more therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, die, in a pharmaceutically-acceptable carrier to the subject in an amount and for a period of time sufficient to treat the deficiency or disorder in the subject suffering from such a disorder.

Thus, the disclosure embraces the delivery of vectors encoding one or more peptides, polypeptides, or proteins, which are useful for the treatment or prevention of disease states in a mammalian subject. Exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors. Other non-limiting examples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TOF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10 (187A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18. In some

In some embodiments, therapeutic proteins include any polypeptide that is suitable for the purpose of delivering the vector and/or treating or preventing a disease.

The vectors disclosed herein may comprise a transgene to be transferred to a subject to treat a disease associated with reduced expression, lack of expression or dysfunction of the native gene. Exemplary genes and associated disease states include, but are not limited to: glucose-6-phosphatase, associated with glycogen storage deficiency type 1A; phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency; galactose-1 phosphate uridyl transferase, associated with galactosemia; phenylalanine hydroxylase, associated with phenylketonuria; branched chain alpha-ketoacid dehydrogenase, associated with Maple syrup urine disease; fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia: medium chain acyl CoA dehydrogenase, associated with medium chain acetyl CoA deficiency; omithine transcarbamylase, associated with omithine transcarbamylase deficiency; argininesuccinic acid synthetase, associated with citrullinemia; low density lipoprotein receptor protein, associated with familial hypercholesterolemia: UDP-glucoaronosyltransferase, associated with Crigler-Najjar disease; adenosine deaminase, associated with severe combined immunodeficiency disease: hypoxanthine guanine phosphoribosyl transferase, associated with Gout and Lesch-Nyan syndrome; biotinidase, associated with biotinidase deficiency; beta-glucocerebrosidase, associated with Gaucher disease: beta-glucuronidase, associated with Sly syndrome; peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; porphobilinogen deaminase, associated with acute intermittent porphyria; alpha-1 antitrypsin for treatment of alpha-1 antitrypsin deficiency (emphysema): erythropoietin for treatment of anemia due to thalassemia or to renal failure; vascular endothelial growth factor, angiopcictin-1, and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitor for the treatment of occluded blood vessels as seen in, for example, atherosclerosis, thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; the beta adrenergie receptor, anti-sense to, or a mutant form of, phospholamban, the sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for the treatment of congestive heart failure; a tumor suppressor gene such as p53 for the treatment of various cancers; a cytokine such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancers; dystrophin or minidystrophin and atrophin or miniutrophin for the treatment of muscular dystrophies; and, insulin for the treatment of diabetes.

The following are further non-limiting examples of gene products (e.g., proteins) that may be encoded by transgenes of the vectors disclosed herein to treat a disease associated with reduced expression, lack of expression or dysfunction of the native gene: a-galactosidase, acid-glucosidase, adiopokines, adiponectin, alglucosidase alfa, anti-thrombin, ApoAV, ApoCII, apolipoprotein A-1 (APOA1), arylsulfatase A, arylsulfatase B, ATP-binding cassette transporter A1 (ABCA1), ABCD1, CCR5 receptor, erythropoietin, Factor VIII, Factor VII, Factor IX, Factor V, fetal hemoglobin, beta-globin, GPI-anchored HDL-binding protein (GPI-HBP) I, growth hormone, hepatocyte growth factor, imiglucerase, lecithin-cholesterol acyltransferase (LCAT), leptin, LDL receptor, lipase maturation factor (LMF) 1, lipoprotein lipase, lysozyme, nicotinamide dinucleotide phosphate (NADPH) oxidase, Rab escort protein-1 (REP-1), retinal degeneration slow (RDS), retinal pigment epithelium-specific 65 (RPE65), rhodopsin, T cell receptor alpha or beta chains, thrombopoeitin, tyrosine hydroxylase, VEGF, von heldebrant factor, von willebrand factor, and X-linked inhibitor of apoptosis (XIAP).